First person – Rebeka Tomasin Free

Rebeka Tomasin

How would you explain the main findings of your paper in lay terms?

Most cells in our body are not actively proliferating but are kept in a non-proliferative state by the surrounding microenvironment and might proliferate again upon appropriate stimuli. It is not easy for cells to overcome the ‘microenvironmental enchantment’ that keeps them in this non-proliferative state (known as ‘quiescence’), which might be one reason why we don't get cancer more often. During puberty, the mammary gland grows and branches. Cells in the invasive leading tips (known as terminal end buds or TEBs) are proliferative, and cells in the ducts behind these TEBs are quiescent and differentiated. This is only possible because cells in ducts and TEBs are exposed to different microenvironments: the basement membrane surrounding the TEBs is laminin poor, thinner and discontinuous, whereas the basement membrane surrounding ducts is laminin rich, continuous and thicker. Using cell culture models of basement membrane-induced mammary gland differentiation, we demonstrated that quiescence has multiple layers of regulation (RNA and protein levels, as well as posttranslational modifications) in several pathways, which is consistent with the resilience of the quiescent state. Interestingly, quiescent cells do receive and sense extracellular proliferative signals, but these signals get shut down, and therefore there is no proliferation. The factor Pten is upregulated in quiescent cells and works as a dam – a disrupting node between upstream signaling and downstream effectors. Inhibition of Pten reactivated proliferation and disrupted tissue architecture in 3D models of mammary gland acinogenesis. Ducts have higher Pten levels than TEBs, which correlated with the thickness of the basement membrane in these structures, and luminal cells in ducts or cells near the nascent lumen in TEBs displayed Pten predominantly polarized to the apicolateral membrane. These findings support a model where physiological acquisition and maintenance of quiescence and differentiation is conferred by a complex network in which Pten plays a crucial role.

Were there any specific challenges associated with this project? If so, how did you overcome them?

Imaging multiple ducts and TEBs in the developing mammary glands, carefully developing a pipeline and analyzing the microscopy data took hundreds of hours. I had to become best friends with the DMi8 and SP8 (the microscopes), and StrataQuest (the analysis software). Also, throughout this project, we have used almost 50 different antibodies, and many of them we received as generous gifts from other labs whom we contacted. Seek and you'll find – even reagents.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

Sometimes little things make all the difference, and we should celebrate them, because they happen more often than breakthroughs. As an example of small daily victories, I could mention when I decided to try antigen retrieval using boiling Tris-EDTA at pH 9 in fresh frozen mammary fat pad sections, and it worked, allowing for Pten staining and all the beautiful images we generated for this paper.

Why did you choose Journal of Cell Science for your paper?

Many of the authors whose research I read have published an original paper in Journal of Cell Science, and I wanted to have one of my own there also. I recently had a Review published here, and it went through a high-level, meticulous revision and editorial process that I've never encountered before, making it a great experience. This particular work looks at mammary gland development from a molecular, cellular and tissue biology standpoint, bringing new hints to how upstream pro-proliferative extracellular signals can be modulated and counteracted downstream in quiescent cells, and to why quiescence is such a resilient phenotype. We thought that our approaches, findings and discussions would then suit Journal of Cell Science, and we are very happy they did. Now I do have my first-author original paper here!

Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?

I've always been very lucky with the people I have crossed paths with, and research is not an exception. I had two major ‘developmental leaps’ in my scientific trajectory.

The first one was spending some time at Robin Anderson's lab in Australia during my PhD. Rick Redvers, the postdoc who supervised me, taught me many of the core things that I now teach other people. It was in the Land Down Under where I found out that, in essence, I am a ‘metastasizer’ of ideas.

The second one was joining Alexandre Bruni-Cardoso's lab at the University of São Paulo. Alexandre is bold in his ideas and approaches, and his door is always open to discuss science. He was the first mentor I ever had that indeed discussed career options with me (I feel this is often neglected during scientific mentorship, and it really shouldn't be). He gave me many opportunities: he would trust and recommend me to give lectures at workshops and meetings on his behalf, and he also made the bridge between me and my current mentor, Cyrus Ghajar. I must also mention Nicolas Hoch, another PI at University of São Paulo, who would often come to my desk or bench where we would talk about data and wild theories, sometimes for hours. These events and these people helped to tailor me as a scientist.

What motivated you to pursue a career in science, and what have been the most interesting moments on the path that led you to where you are now?

The scientist in me has always been there. I wasn't even 9 years old when I created my own ‘lab’ in my backyard. I was the PI, of course, and my brother was my collaborator. We did not have those fancy chemistry kits, but we had snakes that my dad had killed preserved in jars with ethanol. We inspected gecko eggs against the sun to see if the little gecko was ready to hatch. We made soap out of castor beans (and my mum did let us shower with it, despite not using it herself). I would fill up plastic cups with live frogs and bring them to my teacher in first grade. My science teacher in secondary school once interrupted my presentation on ‘gene therapy’ and brought in the principal to watch it. I became a biology and chemistry tutor in high school, and I had to give multiple lectures on DNA, RNA and osmosis because a single room wouldn't fit all the students that came. I knew I would become a biologist.

A great story from my PhD days was when my friend Aline Toneto, also a PhD candidate, came to me for advice. I helped her with a few ‘wrapping up’ experiments and we wrote the paper over a long weekend. That paper turned out to be the highest impact factor paper our PI ever had.

I like to see luck as the combination of skill and opportunity. Being at Bruni-Cardoso's lab, in the most prestigious university in the country, created many opportunities, and through hard work, study and dedication I built up my skills. So, in 2020, when Nicolas Hoch specifically recruited me to work on a COVID-related project, I noticed a distinct staining pattern for ADP-ribose in interferon-treated cells and that expression of the SARS-CoV-2 macrodomain could decrease it. Nicolas recently told me that several projects in his lab have derived from this observation. Right place, right time, right skills.

Who are your role models in science? Why?

Gregor Mendel, for his neat observations that were formative for the field of genetics. Marie Curie – the only person ever to win two Nobel Prizes in two different areas of science – literally gave her life for science. Stephen Paget, the father of the ‘seed and soil’ theory for non-random metastatic dissemination. Judah Folkman, for his pioneering studies in tumor angiogenesis. I am amazed by people that propose and often prove right ideas that make the current ‘truth’ obsolete.

What's next for you?

I cannot picture myself doing anything other than science as a career. Not because I am incapable of doing other things, but because I don't want to. I have recently started as a postdoc in Cyrus Ghajar's lab at the Fred Hutchinson Cancer Center, where I am working on chemotherapy-driven vascular evolution and its effects on the biology of disseminated tumor cells. I feel like my third ‘major career event’ has already begun. Despite being a planner, life sometimes takes unexpected turns that are actually better than our plans and more creative than our imagination. I will keep working hard and enthusiastically to see where this path will take me, keeping in mind the wise words from the Nobel Prize winner T. S. Eliot: ‘The journey, not the destination matters’. And I do love this journey!

Tell us something interesting about yourself that wouldn't be on your CV

I used to bake personalized cakes for my friends – with shapes ranging from ‘E.T. the Extra-Terrestrial’ to a tissue culture flask. They always say ‘if you ever wanted to quit science, you could become a baker’ – but I still would like to keep science as a career and baking as a hobby!